The overall aim of the proposed research will be to develop and characterize structurally, biochemically and mechanically, three-dimensional (3-D) micropatterned cultures of neonatal myocardial cells in which in-vitro cell geometry, alignment and cell-cell contact have been engineered to recapitulate the adult in-vivo phenotype. The structure of 3-D myocyte cultures will be characterized by immunofluorescence confocal microscopy in vitro under resting, paced and mechanically loaded conditions. The contribution of cardiac fibroblasts to the 3-D culture of cardiac myocytes will be characterized and the interaction of these cell types investigated. Novel traction force microscopy techniques will be applied to micro-engineered 3-D myocardial constructs in order to measure their contractile mechanical function. In this way, we hope to gather important information to develop treatment for a leading cause of death in developed nations: chronic congestive heart failure (CHF). Because the only current effective treatment, organ transplantation, is an option only for a small fraction of those affected with CHF, techniques such as cardiac repair, surgical remodeling strategies, and myocardial tissue engineering are becoming more and more important to efficacious treatment. Novel in vitro systems have been designed primarily to investigate basic scientific questions on the molecular and physical determinants of cardiac cellular and extracellular matrix remodeling. In the present proposal, these new methods will be brought together to optimize and characterize biochemically, structurally and mechanically 3-D multi-layered microengineered co-cultures toward the eventual goal of tissue engineering a functional cardiac muscle replacement construct.